Patterning of Diverse Mammalian Cell Types in Serum Free Medium with Photoablation Vipra Dhir NanoScience Technology Center, University of Central Florida, Orlando, FL 32826 Dept. of Mechanical, Materials and Aerospace Engineering, University of Central Florida, Orlando, FL 32826
Anupama Natarajan NanoScience Technology Center, University of Central Florida, Orlando, FL 32826 Burnett College of Biomedical Sciences, University of Central Florida, Orlando, FL 32826
Maria Stancescu NanoScience Technology Center, University of Central Florida, Orlando, FL 32826
Anindarupa Chunder NanoScience Technology Center, University of Central Florida, Orlando, FL 32826 Department of Chemistry, University of Central Florida, Orlando, FL 32826
Neelima Bhargava NanoScience Technology Center, University of Central Florida, Orlando, FL 32826
Mainak Das NanoScience Technology Center, University of Central Florida, Orlando, FL 32826 Burnett College of Biomedical Sciences, University of Central Florida, Orlando, FL 32826
Lei Zhai NanoScience Technology Center, University of Central Florida, Orlando, FL 32826 Department of Chemistry, University of Central Florida, Orlando, FL 32826
Peter Molnar NanoScience Technology Center, University of Central Florida, Orlando, FL 32826 Burnett College of Biomedical Sciences, University of Central Florida, Orlando, FL 32826 DOI 10.1021/bp.150 Published online March 30, 2009 in Wiley InterScience (www.interscience.wiley.com).
Integration of living cells with novel microdevices requires the development of innovative technologies for manipulating cells. Chemical surface patterning has been proven as an effective method to control the attachment and growth of diverse cell populations. Patterning polyelectrolyte multilayers through the combination of layer-by-layer self-assembly technique and photolithography offer a simple, versatile, and silicon compatible approach that overcomes chemical surface patterning limitations, such as short-term stability and low-protein adsorption resistance. In this study, direct photolithographic patterning of two types of multilayers, PAA (poly acrylic acid)/PAAm (poly acryl amide) and PAA/PAH (poly allyl amine hydrochloride), were developed to pattern mammalian neuronal, skeletal, and cardiac muscle cells. For all studied cell types, PAA/PAAm multilayers behaved as a cytophobic surface, completely preventing cell attachment. In contrast, PAA/PAH multilayers have shown a cellselective behavior, promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108-15 cells) to a greater extent, while providing little attachment for neonatal rat cardiac and skeletal muscle cells (C2C12 cell line). PAA/PAAm multilayer cellular patterns have also shown a remarkable protein adsorption resistance. Protein adsorption protocols commonly used for surface treatment in cell culture did not compromise the cell attachment inhibiting feature of the PAA/PAAm multilayer patterns. The combination of polyelectrolyte multilayer patterns with different adsorbed proteins could expand the applicability of this technology to cell types that require specific proteins either on the surface or Correspondence concerning this article should be addressed to P. Molnar at
[email protected]. C 2009 American Institute of Chemical Engineers V
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in the medium for attachment or differentiation, and could not be patterned using the tradiC 2009 American Institute of Chemical Engineers Biotechnol. Prog., 25: tional methods. V 594–603, 2009 Keywords: cell patterning, polyelectrolyte multilayers, layer-by-layer, photolithography, protein adsorption, cell culture
Introduction Manipulation of mammalian cells has attracted a lot of attention because of its potential application in tissue engineering, biosensors and drug screening devices. Numerous methods, including patterning through surface modifications,1 have been developed to generate proper position and interaction of cells. Various approaches, such as UV lithography,2 laser ablation,3 soft lithography4,5 and laminar flow patterning in microfluidic channels,5 and materials, such as photoresists,2 polylysine,3 alkanethiolates,4,6 elastomeric PDMS (polydimethylsiloxane) membrane,7 phospholipid bilayers,8 PEO (polyethylene oxide) terminated triblock copolymer,9 hyperbranched poly(acrylic) acid films,10 grafted polyethylene oxide,11 polyethylene glycol hydrogels,12 polyelectrolyte multilayers,13 interpenetrating network of polyacrylamide and polyethylene glycol,14 polyglycolic acid,15 functionalized poly-p-xylylenes,16 and hyaluronic acid,17 have been successfully used for patterning and cell attachment supporting or inhibiting surfaces. One obstacle, which limits the application of these novel technologies in actual devices, is the relatively short lifetime of the created cellular patterns.13,18,19 In many cases, the patterns are destroyed within a few days after plating, as cells start to grow in the cell resistant areas. Possible causes for this short-term stability of the chemical surface patterns are (1) degradation of the coating material through oxidation or other mechanisms19 and (2) a slow build up of an adsorbed protein layer, originating from the culture medium (serum) or secreted by the cells themselves, on the top of the surface patterns.20–22 In contrast to chemical surface patterns, cell adhesion resistance of polyelectrolyte multilayers is not based on the hydrophobicity of the surface, but on the molecular architecture and physical properties of the film.23 Therefore, they are more resistant to the modifying effect of adsorbed proteins. Moreover, certain polyelectrolyte multilayers, for example, poly acrylic acid (PAA)/poly acryl amide (PAAm) when crosslinked is highly stable24 and their deposition is a simple process, very similar to biological systems with nanoscale control over thickness, compositions, and molecular structure.13 Polyelectrolyte multilayers can be either cell attachment resistive or promoting depending on their properties and the cell type,23,25–27 this makes them promising candidates for patterning diverse cell populations. Although the patterning of several cell types, such as NR6 fibroblast,19 neuron,28–30 primary hepatocytes,31 chondrosarcoma cells,32 microvascular endothelial cells,33 and smooth muscle cells,34 has already been demonstrated using polyelectrolyte multilayers, a comparative study with more than two cell types has not been done. Moreover, the combination of polyelectrolyte multilayers with different adsorbed proteins and the investigation into the protein resistance limits of the patterns could expand the applicability of this technology to cell types which require specific proteins for attachment or differentiation either on the surface or in the medium, and could not be patterned using the traditional methods. This study investigated the applicability of polyelectrolyte multilayers for the
patterning and manipulation of different mammalian cell types. In our experiments, two types of polyelectrolyte multilayers, PAA/PAAm (poly acrylic acid/poly actylamide) and PAA/PAH (poly allyl amine hydrochloride), were used as the patterned substrates, and four cell types, embryonic rat hippocampal cells, neonatal rat cardiac cells, the skeletal muscle C2C12 cell line, and the neuroblastoma/glioma NG108-15 neuronal cell line, were studied. In all cases, the cells were cultured in serum-free medium to prevent the cover up of the surface patterns by proteins generally adsorbed from the serum containing medium.
Materials and Methods Coatings Materials. Poly(acrylic acid) (PAA) (MW 90,000, 25 wt % solution) was purchased from Polysciences. Poly(acryl amide) (PAAm) (MW 10,000, 50 wt % solution) and poly (allyl amine hydrochloride) (PAH) (MW 70,000) were purchased from Sigma Aldrich. Trimethoxysilylpropyldiethylenetriamine (DETA) was obtained from United chemical Technologies Inc. Multilayer Deposition on Coverslips. DETA coverslips were prepared as described by Das et al.35 by cleaning glass coverslips (VWR, 22 22 mm2) with O2 plasma cleaner (Harrick, Ithaca, NY) for 30 min at 100 mTorr. DETA was deposited on clean coverslips by dipping them in 0.1% (v/v) mixture of DETA in freshly distilled toluene. The DETA coverslips were heated to just below the boiling point of toluene for 30 min, rinsed with toluene, and again heated to just below the boiling point of toluene. The coverslips were then dried in an oven overnight. The DETA coverslips were coated with the polyelectrolyte multilayers on an automatic dipping machine (StratoSequence Slide Stainer). A total of 0.01 M solutions of PAA and PAAm were prepared in deionized (DI) water by taking the molecular weight of the repeat unit of each polymer into consideration; in addition, their pH was adjusted to 3.0 using a 1 M HCl aqueous solution. First, the coverslips were immersed in the PAA solution for 15 min and then rinsed with pH 3.0 water three times in separate beakers for 2 min, 1 min, and 1 min, respectively. The coverslips were then immersed in PAAm solution for 15 min and then rinsed with pH 3.0 water three times as described earlier. This cycle was repeated 20 times to deposit 20 bilayers of PAA and PAAm. The coverslips were then kept in an oven at 140 C for 8 h for cross linking. Similarly, PAA/PAH multilayers were deposited by using 0.01 M solutions of PAA and PAH at a pH of 3.5 and 8.5, respectively. The coverslips were immersed in the PAA solution first for 15 min and then rinsed with DI water three times in separate beakers for 2 min, 1 min, and 1 min, respectively. The coverslips were then immersed in the PAH solution for 15 min and then rinsed with DI water three times as described earlier. This cycle was repeated 20 times to deposit 20 bilayers of PAA and PAH. Patterning of the Coverslips by Laser Ablation. The coverslips were patterned using a deep UV (193 nm) excimer
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laser (LambdaPhysik) at a pulse power of 230 mW and a frequency of 10 Hz for 2 min through a quartz photomask (Bandwith Foundry, Eveleigh, Australia). Patterns were visualized by phase contrast microscopy or with an epifluorescent microscope after fluorescent tagging, namely, the PAA/PAAm patterned coverslips were dipped in fluorescently tagged PAH at a pH of 8.5. Cell cultures Materials (a) Cardiac myocyte cuture media and other materials: Calcium and magnesium free Hank’s balanced salt solution (HBSS), trypsin, trypsin inhibitor, collagenase, and Leibovitz medium were obtained from Worthington Biochemical Corporation together in their neonatal cardiomyocyte isolation system. Ultraculture (general purpose medium) was obtained from Bio Whittaker Cambrex. Dulbecco’s modified eagle medium (DMEM) (containing high glucose 1, 4.5 g/L D-glucose, L-glutamine, 110/mg/L sodium pyruvate), L-glutamine (200 mM, 100), penicillin (10,000 units/mL, 100), streptomycin (10,000 lg/mL, 100), B-27, nonessential amino acids (MEM NEAA) (100), HEPES buffer (1 M), and fetal bovine serum were obtained from Gibco/Invitrogen. Dextrose was obtained from Fisher Scientific. Growth factors L-thyroxine and epidermal growth factor (EGF) were purchased from Sigma and hydrocortisone was purchased from BD Biosciences. Human fibronectin was obtained from BD Biosciences. (b) Hippocampal cell culture media: Hibernate E medium was purchased from BrainBits (Springfield, IL). Neurobasal E medium, B27, Glutamax, and antibiotic/antimycotic supplement were obtained from Invitrogen. (c) NG cells and C2C12 cell culture media: DMEM by HyQ (containing 4 mM L-glutamine, 4,500 mg/mL glucose, and sodium pyruvate), fetal bovine serum, HAT (100), and B-27 supplement were obtained from Gibco. Culture Methods (a) Cardiac myocytes: Two-day-old rat pups were euthanized with halothane. Hearts were dissected and minced in ice-cold HBSS. Cardiac myocytes were dissociated by incubation in trypsin (100 lg/mL HBSS) for 20 h at 2–8 C followed by collagenase (300 units/mL L15 medium) treatment for 45 min at 37 C and mechanical trituation. The cell solution was then centrifuged at 50 g for 5 min at 25 C. The cells were resuspended in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin streptomycin and preplated in petridishes and kept in an incubator at 37 C and 5% CO2 for 45 min. The preplating step was carried out to separate fibroblasts from myocytes. The supernatant from the petridishes were centrifuged at 50g for 5 min at 25 C. The cells were resuspended in the plating medium consisting of ultraculture medium supplemented with 9% B-27, 1% Lglutamine, 1% penicillin, 3 mg/ml streptomycin, 1% nonessential amino acids, and 1% HEPES buffer. Growth factors were added in following concentrations adapted from Mohamed et al.36 to the medium, L-thyroxine 0.1 lg/mL, EGF 10 ng/mL, and hydrocortisone 0.5 lg/mL. Cells were plated at a density of 105 cells/cm2 on the coverslips. The medium was changed after 24 h of plating. Subsequent changing of the medium was carried out every fourth day. (b) Hippocampal cells: Embryonic rat hippocampal cells were cultured according to established protocols.37 Briefly, the hippocampus was dissected from E18 rat embryos in icecold Hibernate E medium. Tissue was minced and mechani-
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cally dissociated using a 1 mL pipette. Cells were centrifuged at 300g, 4 C for 2 min and resuspended in the plating medium consisting of Neurobasal E medium supplemented with B27, Glutamax, and antibiotic/antimycotic. Cells were plated at a density of 100 cells/mm2. Cultures were maintained in an incubator at 5% CO2 and 37 C. (c) NG108-15 cells: About 1 million frozen NG 108-15 cells were thawed and centrifuged at 300g for 5 min in NG proliferation medium consisting of DMEM, 2% HAT and 10% FBS. The cells were resuspended in the same medium and plated in a 75 cm2 culture flasks for proliferation. On confluency, the cells were plated on the PAA/PAH patterned coverslips at a density of 100 cells/mm2 in the serum-free differentiating medium consisting of DMEM and 2% B-27. (d) C2C12 cells: C2C12 cells were cultured and plated according to the same protocol as the NG108-15 cells38 on PAA/PAAm patterned coverslips. The coverslips were incubated with different proteins before plating the C2C12 cells. The plating density was 300 cells/mm2. X-ray photoelectron spectroscopy The bare glass, PAA/PAAm, PAA/PAH coatings, Laser ablated PAA/PAAm, PAA/PAH coatings, and protein incubated coverslips were examined with X-ray photoelectron spectroscopy (XPS) using a Kratos (Manchester, UK) Axis 165 equipment according to established protocols.29,35,39 XPS survey scans and high-resolution C 1s, N 1s, O 1s, and Si 2p were obtained using monochromatic Al ka excitation. Contact angle measurement Contact angle measurements were performed according to published protocols.35,39 Briefly, contact angle of a static, sessile drop (5 lL) of DI water was measured using a CAM 200 digital goniometer (KSV Instruments, Ltd.). Three measurements were taken and averaged. Statistical analysis of the cell resistance of the coatings The cell resistance and cell adhering properties of the coatings were evaluated statistically by plating all the above cell types on plain glass coverslips and coverslips coated with PAA/PAH and PAA/PAAm. The cells were counted in each frame at a magnification of 10 and the number of cells observed at 10 magnification were averaged over 10 frames for two coverslips of each type. The counting was done on Days 3 and 6 of the culture and number of cells observed per unit area was reported. Patch clamp electrophysiology Materials. All chemicals were purchased from Sigma Aldrich. Borosilicate glasses (BF150-86-10) were obtained from Sutter (Novato, CA). Patch Clamp Method. Patch clamp experiments on hippocampal and cardiac cells were performed according to published protocols.35,39 In brief, whole-cell patch clamp recordings were performed in a recording chamber on the stage of a Zeiss Axioscope 2FS Plus upright microscope at room temperature, in the culture medium, where the pH was adjusted to 7.3 with HEPES. Patch pipettes were prepared from borosilicate glass with a Sutter P97 pipette puller and filled with intracellular solution (in mM: K-gluconate 140, EGTA 1, MgCl2 2, Na2ATP 2, Hepes 10; pH ¼ 7.2). The
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Figure 1. XPS survey scans of (A) clean glass, (B) PAA/PAAm coating, (C) ablated PAA/PAAm, (D) ablated PAA/PAAm after fibronectin incubation, (E) PAA/PAH, (F) Ablated PAA/PAH.
resistance of the electrodes was 6–8 MX. Voltage clamp and current clamp experiments were performed with a Multiclamp 700A amplifier (Axon, Union City, CA). Signals were filtered at 3 kHz and digitized at 20 kHz with an Axon Digidata 1322A interface. Data recording and analysis were performed with pClamp 10 software (Axon). Action potentials were evoked with 1 s depolarizing current injections from a 70 mV holding potential.
Results Polyelectrolyte deposition, patterning, and visualization After several trials, we observed that for the initialization of the first layer of the polyelectrolyte, DETA covalently – modified glass coverslips had clear benefits compared with clean glass as the substrate. DETA coverslips had a strong
inherent positive charge on the surface, which made the uniform deposition of the multilayers easier in comparison with deposition onto clean glass substrates. Formation of polyelectrolyte multilayers were verified by contact angle measurements, XPS, and visual inspection. Static contact angle values for PAA/PAAm and PAA/PAH multilayers were 101.4 5.2 and 67.1 3.3 , respectively, which indicated the presence of the polymer on the surface as compared with glass that got completely wet forming a 0 contact angle. The appearance of large carbon and nitrogen peaks in the XPS spectra verified the presence of uniform polymer film on the surface. The ablation time for patterning the polyelectrolyte multilayers was set to remove all measurable traces of the film, which was proven by XPS measurements on the coated and ablated coverslips. The XPS survey spectra obtained on the
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Figure 2. Visualization of patterned polyelectrolyte multilayers by (A) phase contrast microscopy and (B) fluorescently tagged PAH. The arrows depict ablated region. Scale bar depicts 100 lm.
Table 1. Statistical Analysis of the Cell Attachment Promoting Effect of the Surfaces Surface
Cardiac
C2C12
(A) Statistical analysis, cells/view n ¼ 3, error represents SD, Day 3 of the culture. PAA PAAm 9.6 5.5 00 PAA PAH 55.5 25 51 10.5 Glass 418.5 75 122 21.5 (B) Statistical analysis n ¼ 3, error represents SD, Day 6 of the culture. PAA PAAm 6.2 5.5 00 PAA PAH 133 52 18.5 11 Glass 447 83 157.5 30.5
glass substrate, after the deposition of PAA/PAAm multilayers and after ablation of the film, are shown in Figure 1. The carbon and nitrogen peaks, characteristics of the PAA/PAAm film, were observed on the PAA/PAAm coated glass substrates, but not on the bare glass substrates or the PAA/PAAm coated glass substrates followed by laser ablation. Similarly, large carbon and nitrogen peaks were observed on PAA/PAH modified coverlips, whereas after ablation, there were only traces of carbon present and nitrogen was totally absent. After selective ablation of the PAA/PAAm multilayers through a photomask (patterning), the border between the ablated and nonablated regions were clearly visible through a standard phase-contrast microscope (Figure 2A). For a more reliable visualization of the electrically charged multilayers, fluorescently tagged PAH was used to bind with the multilayers and make the films fluorescent (Figure 2B). Patterned PAA/PAAm multilayers were dipped into fluorescently tagged PAH at pH 8.5. It has been shown by Choi et al.40 that at pH 8.5, 50% PAH is protonated making it positively charged. They have also reported that when PAA is assembled at pH 3 with either an uncharged or fully charged polyelectrolyte it is 30% ionized. Therefore, it can be assumed in case of PAA/PAAm assembled at pH 3, 30% of PAA was ionized leaving only 70% of PAA to form hydrogen bonds with PAAm. This hydrogen bonded PAA/PAAm later forms imide bond on crosslinking. The 30% left PAA with out the imide bond, when at pH 8.5 gets ionized and attached to the protonated fluorescently tagged PAH, making the PAA/ PAAm multilayers visible under fluorescence. According to Yang et al.,24 the stability of the polyelectrolyte multilayers PAA/PAAm was greatly enhanced because of the formation of imide bond by crosslinking between PAA and PAAm when exposed to 140 C for 8 h. Therefore, we also used same thermal crosslinking, to stabilize PAA/
Hippocampus
NG 108-15
00 125 31 19.5 11.1
00 138.5 32.4 84
00 96.5 26 8.5 6
00 95.5 27 5.5 9
Table 2. Cell Attachment Behavior of Polyelectrolyte Multilayers and Glass Cell Type
PAA/PAAm
PAA/PAH
Glass
Cardiac myocytes Hippocampus cells NG 108-15 C2C12
Resistant Resistant Resistant Resistant
Adhesive Adhesive Adhesive Adhesive
Adhesive Resistant Resistant Adhesive
PAAm coating at physiological pH so that it can be used at pH of cell culture medium. Cell attachment and growth on polyelectrolyte multilayers and patterns Cell patterning usually requires two types of surfaces: one promotes cell attachment and growth, whereas the other prevents cell attachment and growth. To establish the polyelectrolyte multilayers PAA/PAAm and PAA/PAH as true cytophobic and cytophilic surfaces, respectively, statistical analysis on the cell attachment behavior of polyelectrolyte multilayers was carried out. The statistical data on the attachment of the various cell types on the different surfaces are provided in Table 1. The number of cells attaching to plain glass, PAA/PAAm and PAA/PAH surface/mm2, were measured and compared. From Table 1 it can be seen that for all studied cell types, the PAA/PAAm multilayer behaved as a cell resistant surface, completely preventing cell attachment and growth. In contrast, the PAA/PAH multilayer showed a cell-selective behavior by promoting the attachment and growth of neuronal cells (embryonic rat hippocampal and NG108-15 cells) to a greater extent and, to some extent, skeletal muscle cells and neonatal rat cardiac cells. A summary of different surfaces and their cell attachment behavior is given in Table 2.
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Figure 3. Cell attachment and growth on patterned polyelectrolyte multilayers PAA/PAAm was negative for all cell types. PAA/PAH was positive for neurons and negative for cardiac myocytes and skeletal muscle cells. Glass was negative for neurons, but allowed attachment and growth of cardiac myocytes and skeletal muscle cells. (A,B) Cardiac myocytes and C2C12 cells attached to ablated region, i.e., glass and not PAA/PAAm, (C,D) neurons hippocampal cells and NG cells attached to PAA/PAH and not to be ablated region, i.e., glass. Scale bar depicts 100 lm.
Unfortunately, we were not able to use a second polyelectrolyte multilayer to backfill the ablated patterns, as it is common with self-assembled monolayers, to enhance the contrast between cell growth enabling and resisting areas. For this a clean glass was used as the alternative surface for cell patterning. Glass was utilized as a positive surface for cardiac myocytes and C2C12 skeletal muscle cells with PAA/PAAm negative background; however, it was used as the negative surface for neurons with PAA/PAH as the positive surface. The patterns of the cells on PAA/PAAm and PAA/PAH multilayers are shown in Figure 3. However, glass is not an ideal negative surface for promoting physiological development of certain cell types. For example, as reported earlier,38 C2C12 skeletal muscle cells do not form myotubes in serum-free medium without contact signaling that originated from the growth surface. Based on the fact that the attachment of cardiac myocytes is significantly better with fibronectin or serum on the surface, we have taken advantage of the remarkable protein adsorption resistive feature of polyelectrolyte-based cellular patterns and used protein modified patterns to promote cell growth.
tions of time before cell plating. As noted in Figure 4A, cardiac myocytes were complying with the patterns even after 1 h incubation in 0.2 g/L human plasma fibronectin solution. XPS data (Figure 1) show that a layer of fibronectin was adsorbed to the ablated portion of the PAA/PAAm coverslips during this time. This process of incubation of the PAA/ PAAm multilayer with fibronectin did not change its cellattachment and growth resistive properties. In the case of the C2C12 cells, protein absorbed on the glass significantly improved the cell growth and differentiation promoting properties of this surface, enabling the formation of C2C12 myotubes. As shown in Figure 4B, no myotube formation took place in the absence of incubation with 10% serum containing medium. In the experiments presented in Figure 4C,D, the patterned PAA/PAAm coverslips were incubated with a 10% serum containing medium (NG proliferation medium) for different time periods. After completion of the incubation time C2C12 cells were seeded on the patterns. The patterns were observed on the second day of seeding the cells. The pictures show that the pattern was formed without the protein incubation, but the formation of myotubes took place only after incubation with the serum containing medium.
Cell growth on protein—modified polyelectrolyte patterns To assess the protein adsorption resistance of polyelectrolyte-based cellular patterns, we incubated the polyelectrolyte patterns in protein containing solutions for different dura-
Physiology of patterned cells Visual inspection revealed no obvious morphological difference between the cells grown on the polyelectrolyte
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Figure 4. Effect of protein incubation on the patterns and cell morphology. (A) Cardiac myocytes on glass vs. PAA/PAAm surface patterns incubated with fibronectin, Day 17, (B) C2C12 cells did not form myotubes without incubation with serum containing medium, (C,D) C2C12 cells plated on glass vs. PAA/PAAm surface patterns incubated with 10% serum containing medium for 1–8 h, Day 2. Scale bar depicts 100 lm.
patterns and the cells grown on traditional control surfaces (PDL (poly-d-lysine) and DETA for neurons, fibronectin for cardiac myocytes). Hippocampal cells were grown on PAA/ PAH surfaces as a positive with clean glass as the background negative surface. Cardiac cells were cultured on fibronectin-treated clean glass as the positive and PAAPAAm as the negative surface. To evaluate the physiological properties of the excitable cells, whole-cell patch clamp recordings in current clamp mode of spontaneous or evoked action potentials were performed in cardiac and hippocampal cells (Figure 5). For hippocampal cells, repetitive firing was evoked by 1 s current injection. Cardiac cells were spontaneously active; thus, no current was injected. More than 90% of the recorded hippocampal cells fired repetitive action potentials on prolonged depolarization, which is characteristic of mature pyramidal cells in culture. All recorded cardiac cells fired spontaneous, short-action potentials, which is characteristic of mature postnatal rat cardiac cells.35 Long-term stability of the pattern The advantage of using the polyelectrolyte multilayers over other materials for patterning cells is the long-term stability of the patterns, i.e., the cells remain in the cell adhesive areas and
do not over grow in the cell resistant areas which are PAA/ PAAm multilayers. In our studies, polyelectrolyte-based cellular patterns were much more stable than the self-assembled monolayer-based patterns reported earlier where the patterns were only stable up to 1–2 weeks.38 Figure 6 illustrates highfidelity cardiac myocyte patterns after 100 days in culture, which was not achievable with our earlier patterning methods. Also the myocytes were beating for at least 100 days on the pattern. Moreover, our hippocampal patterns were stable for up to 20 days.
Discussion In this study, PAA/PAAm and PAA/PAH polyelectrolyte multilayers were patterned by laser ablation through a photomask to create cell attachment resistive and promoting areas on glass coverslips. The patterns were visualized by simple phase contrast microscopy or fluorescence contrast after the fluorescent tagging. PAA/PAAm multilayers prevented the attachment of all studied cell types. PAA/PAH was cell attachment promoting for embryonic rat hippocampal and NG108-15 cells to a greater extent and somewhat for neonatal rat cardiac myocytes and C2C12 skeletal muscle cells. Cellular patterns on the polyelectrolytes were exceptionally
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Figure 5. Electrophysiological characterization of hippocampal (A) and cardiac cells (B) grown on polyelectrolyte patterns for 2 weeks. Repetitive firing was evoked in hippocampal cells by injection of 10–50 pA current for 1 s in current clamp mode. In cardiac cells, spontaneous action potentials were recorded without current stimulation. In both cell types, membrane potential was hyperpolarized to 70 mV with a holding current before the experiment.
Figure 6. Long-term stability of the polyelectrolyte-based patterns. A: Neonatal cardiac myocytes Day 100 of the culture. B: Hippocampal cells Day 20 of the culture. Scale bar depicts 100 lm.
stable; cardiac myocytes did not overgrow the patterns and were beating for at least 100 days. Cellular patterns created with PAA/PAAm multilayers as the negative surface showed remarkable protein resistance, they tolerated standard, cell culture surface treatment protein adsorption protocols. The layer-by-layer deposition of the polyelectrolyte multilayers was a simple, reliable process, and did not require complex chemical procedures. In comparison with the commonly used covalent surface modification methods, it was simpler, less variable, robust, and stable after crosslinking in cell culture conditions. Another advantage of the polyelectrolyte multilayers was the improved visualization; surface patterns were visible under a normal phase contrast microscope. In specific applications, such as time-lapse imaging or repetitive multilayer patterning, visualization of the patterns has been a challenging requirement. Cell attachment inhibiting or promoting features of polyelectrolyte multilayers was determined by the molecular architecture and the physical properties of the layers. Therefore, the tunable and flexible properties of polyelectrolyte
multilayers can be used to selectively pattern different cell types. Polyelectrolyte patterns combined with a simple and widely used protein adsorption surface treatment, which does not compromise the cell resistance of the background, could significantly enhance cell selectivity, as well as cell attachment promoting and physiological effects of the foreground. These unique properties could lead to various applications in many cell culture laboratories. The origin of the cell resistance of the PAA/PAAm multilayers was based on the physical properties of the layers not the chemical properties. The high degree of swelling (about 3.5 times) of PAA/PAAm in PBS, with the same ionic strength as the cell culture medium, makes the coatings soft and water like that they do not provide a rigid support for cell attachment.13 PAA/PAH coatings deposited at the pH of 3.5/7.5, respectively, have been reported to be cell adhesive as they swell only to 130% of their original thickness in the buffered conditions.23 Laser ablation through a photomask has proved to be a simple and effective way to create polyelectrolyte surface
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patterns; this method was high-throughput and compatible with standard silicon manufacturing process. Technical difficulties prevented the ‘‘backfill’’ of the ablated areas with a second/different polyelectrolyte multilayer, instead, protein modification of the background clean glass was utilized, as it is widely used in most cell culture practices.
Conclusions Photolithographic patterning of polyelectrolyte multilayers is a simple, versatile, and robust method to pattern cells with exceptional long-term stability and protein adsorption resistance. High fidelity beating patterns of neonatal rat cardiac myocytes were observed after 100 days on polyelectrolyte patterns in our serum-free medium. Premade polyelectrolyte patterns combined with commonly used protein adsorption, surface modification methods could extend the applications of the patterned cultures.
Acknowledgment This work was supported by NIH Career Development Award K01 EB003465 and UCF internal funds.
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